The objective of this proposal is for the principle investigator to gain first hand experience in the preparation of a cDNA library, the isolation and sequence analysis of clones from the library, and in the extraction and analysis of RNA from tissues. The proposed experiments are directly relevant to the principle investigator's research into the breakdown and inactivation of neuropeptides which regulate digestion. Biologically active neuropeptides, released from neurones in the wall of the digestive tract, make a major contribution to control of digestion. Peptidase enzymes degrade the neuropeptides soon after secretion and thereby limit the extent and duration of their influence. Therefore, peptidases have the potential to play an important role in regulation. Elucidation of the precise physiological role of these enzymes will require firstly, the identification and chemical characterization of peptidases, and secondly the examination of the control mechanisms which regulate expression of peptidases in health and disease. In this proposal, attention will be directed to two peptidases, a novel carboxypeptidase recently discovered and isolated from human stomach by the principle investigator, and endopeptidase EC3.4.24.11, a well characterized peptidase which degrades a wide range of neuropeptides. There are three specific aims: 1) preparation of a cDNA library from human gastric mucosa; 2) isolation and sequence analysis of clones from the cDNA library which encode for a novel carboxypeptidase recently isolate from human stomach; and 3) examination of the regulation of expression of endopeptidase-24.11 in the rat stomach. Results from the proposed experiments will provide new information about the structure and regulation of neuropeptide degrading enzymes in the stomach and may lead to the development of therapeutically valuable peptidase inhibitors and long acting, enzyme resistant peptides. The results will be incorporated into a competitive renewal of a grant entitled "Peptide Metabolism in Human Stomach." Experience gained will be used to derive the primary structure of other novel peptidases from the stomach and to examine the regulation of peptidase expression in the digestive tract.